System for separating particulate substances by reorienting gradients



March 29, 1966 N. G. ANDERSON 3,243,105

SYSTEM FOR SEPARATING PARTICULATE SUBSTANCES BY REORIENTING GRADIENTS Filed NOV. 15, 1963 ROTOR AT REST ACCELERATING Fig. 3. Fig. 4. Fig. 5.

ROTOR AT SPEED DECELERATING ROTOR AT REST Fig. 6. Fig. 7. I Fig. 8.

INVENTOR.

Norman 6. Anderson ATTORNEY.

tion.

United States Patent Ofifice Patented Mar. 29, 1966 3,243,105 SYSTEM FOR SEPARATIWG PARTICULATE SUB- STANCES BY REORIENTING GRADIENTS Norman G. Anderson, Oak Ridge, Tenn., assignor to the United States of America as represented by the United States Atomic Energy Commission Filed Nov. 15, 1963, Ser. No. 324,139 3 Claims. (Cl. 233-1) This invention relates to a process and apparatus for separating a heterogeneous colloid sample into its components by centrifugation and more particularly to the use of reorientation of the gradient in a liquid density gradient centrifugation type of separation.

Heretofore it has been the practice in the prior art to establish a centrifugally-stabilized density gradient of the liquid medium under dynamic conditions in an ultracentrifuge. Difiiculties arise with seals, skimmers and other mechanisms associated with liquid transfer as a result of interference with rotor balance, and with the intricacies of construction when liquids must be transferred into and out of a centrifuge bowl under dynamic Conditions.

Applicant, with a knowledge of the problems of the prior art, has for an object of his invention the provision of a process and system for accomplishing the insertion of a liquid density gradient and the sample material into a cetrifuge and the recovery of the separated components of a centrifuged sample both under static conditions.

Applicant has as another object of his invention the provision of a system for the centrifugal separation of particulate components of a colloid sample through the use of an originally gravity-stabilized gradient.

Applicant has as a still further object of his invention the provision of a simplified process for the separation of the components of a particulate colloid sample in a centrifuge by first setting up a vertical gradient with the sample therein, orienting the gradient to horizontal to separate the components of the sample, and then reorienting the gradient to vertical to recover the components.

Other objects and advantages of my invention will appear from the following specification and the accompanying drawings, and the novel features thereof will be particularly pointed out in the annexed claims.

In the drawings, FIG. 1 is a schematic in sectional elevation of a prior art centrifuge for separating components of a liquid. FIG. 2 is a schematic of a conventional mixer for producing vertical gradient liquids. FIG. 3 is a schematic of a rotor of an ultracentrifuge containing a liquid with a vertically-oriented, gravity-stabilized density gradient, including a sample positioned therein. FIG. 4 is a schematic showing the reorientation of the gradient during acceleration of the rotor. FIG. 5 is a schematic showing the gradient orientation after further accelera- FIG. 6 is a schematic showing the centrifugallystabilized gradient when the rotor is at full speed. FIG. 7 is a schematic showing the return to gravity stabilization during deceleration. FIG. 8 is a schematic showing the completely gravity-stabilized gradient with the rotor at rest, and after separation of the particulate components of the sample.

Referring to the drawings in detail, FIG. 1 is represena-tive of the centrifuges of the prior art. In its use a density gradient is established within the rotor 2 spinning at low speed, e.g., 3000 r.p.m., by admitting liquid through a face seal (not shown) through an axial conduit (not shown) of the rotor core 1, and through radial inlet conduits 3, 4 to the rotor periphery. Low density liquid is admitted first, the density gradually increasing until the rotor bowl is completely filled. If a simple,

density-equilibrium method is preferred, the particulate sample is homogeneously premixed into the gradient liquid during the preparation of the gradient. If separation is to involve differential sedimentation rates of the particulate components the sample is admitted through conduit 5 into the low-density end of the gradient in the axial region of the rotor bowl after the filling of the bowl with gradient liquid. This involves reversal of the flow into the bowl 2 by pumping through the seal (not shown). The liquid with vertical gradient may be prepared in a conventional mixer of the type of FIG. 2 and fed to the centrifuge 7 through the spout 8. As an example, sugar solution, a relatively heavy solution, in vessel 10, and water, a relatively lighter solution, in vessel 9, are subjected to the action of weights of conical shape, as shown, as they are lowered into vessels 9 and 10. Initially more of the water flows through line 14 to join a smaller quantity of sugar solution flowing through line 13 to mix and pass through spout 8 to the centrifuge 7. Later the sugar solution will predominate over the water in the mixture. Since the lighter liquid flows into the bottom first and is displaced upwardly by fluid of increasing density, a gradient is gradually formed with the most dense fluid at the bottom.

Referring now to FIG. 3, the liquid density gradient is introduced, low density end first, into the bowl 7 of an ultracentrifuge by suitable conduit means leading to the bottom of said bowl whereby the gradient assumes its position according to vertical orientation and gravity stabilization under static conditions.

The rotor is slowly accelerated, during which the force exerted upon the liquid at any point, at any time, is a resultant in direction and magnitude of the interaction of the gravitational and centrifugal forces as shown in FIG. 4. This causes the density gradient, which initially decreased vertically upward, to undergo a reorientation as shown in FIG. 5. Liquid having a given density value will now be distributed within the liquid according to an upwardly concave paraboloid. Finite differences in density can, thus, be represented by a sequence of imaginary, concentric paraboloids.

At very high angular velocity the force of gravity is small in comparison to the centrifugal value; the highest density liquid will occupy the peripheral region; and the lowest density liquid will occupy the axial region, as shown in FIG. 6. Thus, a radially oriented density is established corresponding to that used in the prior art method comprising establishing the gradient under dynamic conditions.

Operation of the rotor under these conditions causes the particulate sample components to sediment at differential rates and to assume distribution within the gradient according to the known principles of density gradient centrifugation as shown in FIG. 6.

Gradual deceleration of the rotor causes the radial gradient to be reoriented in the manner of FIG. 7, in the reverse sequence of configuration change as described above, to assume the vertical gradient position of FIG. 8, as the centrifugal force diminishes to zero and stability of the gradient is once again maintained by gravitational force alone.

Deformation occurring at the various levels may be best understood by describing the changes occurring in layers originally at the top, middle, and bottom of the rotor. The fluid originally against the upper rotor cap (not shown) becomes squeezed into a small paraboloid of revolution during acceleration, as shown in FIG. 4, and then occupies the center of the rotor at high speed, as shown in FIG. 6. A zone, X, in the middle of the rotor of FIG. 3, shown at rest, increases in area during acceleration, and then decreases in area slightly as an approximately vertical position is approached. The zone B at the bottom of the rotor, shown at rest in FIG. 3, decreases markedly in surface area during acceleration as shown in FIG. 4, but covers the entire surface of the rotor wall at high speed, as shown in FIG. 6. The greatest area changes, therefore, occur in those zones near the top and bottom when at rest but near the center and the edge at high speed.

The oriented gradient, before the particles have sedimented appreciably, is shown in FIG. 6, and after sedimentation in FIG. 8. The distribution during deceleration is shown in FIG. 7, with the distribution at rest shown in FIG. 8. The separated zones are recovered by draining the gradient out of the bottom of the rotor, or displacing it out the top.

A mathematical analysis of the areas of isodensity surfaces shows that very little shearing occurs in the center of the gradient in a conventional sector-compartmented rotor. While increases and decreases in area occur, the difference in rate of increase or decrease in the areas of adjacent zones is rather small. By placing a dense cushion B in the bottom and an overlay T of light fluid above the sample layer at the top, the sample layer and density gradient may be restricted to that part of the rotor where least shearing occurs. As the fluid layers change position during acceleration and deceleration, their tangential velocity will change, since the velocity at any point in the rotor is a function of both the rotatational speed and the radius of the point. Fluid in the upper layer, originally near the edge of the rotor, decreases in tangential velocity relative to previously underlying fluid during acceleration, for example. Conventional vertical septa (not shown), therefore, may be necessary to prevent swirling during reorientation of the gradient.

When the rotor is brought to rest, various zones may be recovered by successively draining the rotor contents out of the bottom, or by displacing the gradient out through the top.

Having thus described my invention, I claim:

1. A method of separating the particulate components of a sample according to density and sedimentation rate comprising the steps of introducing a liquid having a density gradient into the rotor of a bowl type centrifuge under static conditions, placing a colloid sample having 4 particulate components of different densities or sedimentation rates to be classified and separated in the liquid, centrifuging the liquid and sample to separate the particulate components of the sample, then successively draining portions of the mixture from the centrifuge to recover the separated components of the sample.

2. A method of separating the particulate components of a colloid sample according to their respective densities and sedimentation rates comprising the steps of introducing a liquid having zones of different density into a centrifuge under static conditions to provide a vertical gradient, placing a sample having particulate components of different densities or sedimentation rate, centrifuging the sample and liquid mixture to set up a horizontal gradient and to classify and separate the components of the sample, bringing the mixture to rest to re-establish a vertical gradient, and successively removing the different zones of the mixture from the centrifuge to recover the particulate components of the sample.

3. A method of separating the components of a sample according to their respective densities or sedimentation rates comprising the steps of introducing a liquid having zones of different density into a centrifuge rotor bowl under static conditions to provide a vertical density gradient, placing a sample having particulate components of different sedimentation rates and densities in the liquid, centrifuging the sample and liquid mixture to reorient the density gradient to set up a horizontal gradient and to separate the components of the sample, bringing the mixture to rest to re-establish a vertical gradient, and successively draining the different density zones of the liquid from the bottom of the centrifuge to recover the components of the sample.

References Cited by the Examiner UNITED STATES PATENTS 3,075,694 1/1963 Anderson 233-40 FOREIGN PATENTS 4,308 11/1949 France.

M. CARY NELSON, Primary Examine).

HENRY T. KLINKSIEK, Examiner. 

1. A METHOD OF SEPARATING THE PARTICULATE COMPONENTS OF A SAMPLE ACCORDING TO DENSITY AND SEDIMENTATION RATE COMPRISING THE STEPS OF INTRODUCING A LIQUID HAVING A DENSITY GRADIENT INTO THE ROTOR OF A BOWL TYPE CENTRIFUGE UNDER STATIC CONDITIONS, PLACING A COLLOID SAMPLE HAVING PARTICULAR COMPONENTS OF DIFFERENT DENSITIES OR SEDIMENTATION RATES TO BE CLASSIFIED AND SEPARATED IN THE LIQUID, CENTRIFUGING THE LIQUID AND SAMPLE TO SEPARATE THE PARTICULATE COMPONENTS OF THE SAMPLE, THEN SUCCESSIVELY DRAINING PORTIONS OF THE MIXTURE FROM THE CENTRIFUGE TO RECOVER THE SEPARATED COMPONENTS OF THE SAMPLE. 